Opioids are widely used for pain m management but one unwanted side effect of acute opioid administration is rapid eye movement (REM) sleep inhibition. Pain itself disrupts sleep, and sleep loss is the most common complaint of acute pain patients. Pontine choilnergic neurotransmission is a major contributor to REM sleep generation, and new data suggest that pontine GABAergic transmission and basal forebrain cholinergic transmission participate in REM sleep regulation. The long-term objectives of this renewal application are to advance scientific knowledge by specifying the cellular and molecular mechanisms through which opioids inhibit REM sleep. The four aims are unified conceptually and related to the long-term goals by focusing on opioid modulation of transmitter release and guanine nucleotide binding protein (G protein) activation. These studies focus on brain stem and forebrain regions known to regulate sleep and breathing. Novel aspects include an emphasis on the interactions of opioids with multiple transmitter systems in multiple sleep-related brain regions. Aims 1, 2, and 3 will use in vivo microdialysis and high performance liquid chromatography. Opioids will be administered systemically and, in separate experiments, directly into brain regions of interest by reverse dialysis. Concentration-response and antagonist blocking studies will evaluate receptor mediation of opioid effects. Aim 4 will use in vitro [35S]GTPgammaS autoradiography to examine the interaction of opioids with other sleep-modulatory neurotransmitters at the level of G protein activation. Aim 1 will test the hypothesis that opioids decrease acetylcholine (ACh) release in medullary hypoglossal nucleus. Aim 2 will test the hypothesis that basal forebrain opioid administration decreases basal forebrain ACh release. The Aim 3 hypothesis is that opioids decrease gamma aminobutyric acid (GABA) release in REM sleep-regulating regions of the pontine reticular formation. Aim 4 will test the hypothesis that opioids alter G protein activation by muscarinic cholinergic agonists and by the novel hypothalamic peptide hypocretin-1/orexin-A. Aim 4 will specify whether combined agonist activation of G proteins in sleep-related nuclei is fully additive, partially additive, or non-additive. Aim 4 will permit inferences concerning activation of independent or common G protein pools by mu opioid, muscarinic cholinergic, and hypocretin/orexin receptor agonists. The potential health relatedness of these basic studies derives from the unwanted side effects of opioids on sleep and breathing.